1. Introduction
Chronic pain conditions are among the most common causes of disability worldwide.28 In addition to pain and disability, chronic pain is associated with emotional and cognitive disorders which further decrease quality of life.4,10,25,44,48 One reason why chronic pain is so difficult to treat is that it is associated with persistent anatomical and functional changes and altered gene expression throughout the neuroaxis.1,3,5 Among the brain structures implicated in chronic pain conditions, the prefrontal cortex (PFC) is important in affective, motivational and cognitive functions, and plays a critical role in pain control.34,37,67 Improved understanding of the mechanisms underlying chronic pain–induced neuroplasticity will support development of new therapeutic strategies _targeting these processes.
Epigenetic mechanisms, including DNA methylation, regulate gene expression without affecting DNA sequences,23,24,27 and may play an important role in chronic pain.18,40 Until recently, DNA methylation was regarded as fairly static. However, it is now clear that epigenetic processes are dynamically regulated in the adult nervous system, and that they are crucial for synaptic plasticity and memory formation.57 We previously demonstrated a link between DNA methylation and chronic back pain in murine models and in humans suffering from chronic back pain.65 In addition, peripheral nerve injury is associated with wide-spread and long-lasting changes in DNA methylation in the frontal cortex and T cells in rats,43 and with transcriptome-wide changes1 and decreased global DNA methylation66 in the frontal cortex in neuropathic mice. Interestingly, environmental enrichment reversed both nerve injury–induced hypersensitivity and decreased global DNA methylation in the mouse brain.66,69
DNA methylation is catalyzed by DNA methyltransferases that transfer the methyl group from S-adenosylmethionine (SAM), the principal methyl donor, to the fifth position of cytosine (5-methylcytosine, 5-mC) on the DNA strands.36,42 While the functional consequences of DNA methylation are complex, in general high levels of methylation in 5′ regulatory regions reduce the expression of that gene.64 Although SAM is marketed as a nutritional supplement for a wide range of conditions including depression, cognitive deficits, migraine, back pain, osteoarthritis, and liver support,33,47,52,55 little is known about its efficacy or potential side effects.31,58
Our previous data have shown extensive changes in the DNA methylation of individual genes and an overall decrease in DNA methylation in the rodent frontal cortex after chronic neuropathic pain.43,66 We therefore hypothesized that the methyl donor, SAM, will have therapeutic effects on pain and associated emotional and cognitive comorbidities in a chronic model of neuropathic pain in mice. In this study, emphasis was placed on modeling the use of SAM as a nutritional supplement: (1) treatment was delayed for 3 months after initiation of neuropathic pain to ensure the phenotype was fully established, (2) SAM was given orally as is typical with a supplement, and (3) SAM was administered 3 times a week for an additional 4 months. This design allows for translation of this preclinical study to clinical utility.
2. Materials and methods
2.1. Animals
Male CD1 mice (Charles River Laboratories, St-Constant, QC, Canada) were housed on a 12 hours light/dark cycle in a temperature-controlled room in groups of 3 to 4 in ventilated polycarbonate cages (Allentown, Allentown, NJ), with corncob bedding (7097, Teklad Corncob Bedding, Envigo, United Kingdom) and cotton nesting squares for enrichment. Mice were given access to food (2092X Global Soy Protein-Free Extruded Rodent Diet, Irradiated) and water ad libitum. Before all experimental protocols, animals were habituated to the housing conditions for 1 week.
All experiments were approved by the Animal Care Committee at McGill University and conformed to the ethical guidelines of the Canadian Council of Animal Care and the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.73,74
2.2. Neuropathic pain model: spared nerve injury
Animals were randomly assigned to receive either spared nerve injury (SNI) (n = 15) or sham surgery (n = 12) at 10 to 12 weeks of age. Spared nerve injury surgery consisted of ligation and transection of the left tibial and common peroneal branches of the sciatic nerve under isoflurane anesthesia, while sparing the sural nerve.17 The tibial and common peroneal branches were tightly ligated with 4 to 0 silk (Ethicon) and sectioned distal to the ligation. Sham surgery consisted of exposing the nerve without damaging it.
2.3. Behavioral measures
Baseline behavioral assessments were performed 3 months after SNI or sham surgery to confirm the development of neuropathic pain. Mechanical and cold sensitivity and motor capacity were then reassessed every 2 weeks during the 4 months of treatment. Cognitive behaviors and pain avoidance were assessed at the end of the 4 months of treatment.
2.3.1. Measurement of mechanical and cold sensitivity
After a habituation period of 1 hour to the testing Plexiglas boxes placed on a grid, von Frey filaments (Stoelting Co, Wood Dale, IL) were applied to the plantar surface of the hind paw to the point of bending for 3 seconds or until withdrawal, and mechanical sensitivity was determined as the 50% withdrawal threshold using the up-down method.14 The stimulus intensity ranged from 0.04 to 4.0 g, corresponding to filament numbers 2.44 to 4.56. Cold sensitivity was assessed using a modified version of the acetone drop test16: after an application of 25 μL of acetone to the plantar surface of the hind paw, the total duration of acetone-evoked behaviors (flinching, liking, and biting) was measured for 1 minute.
2.3.2. Measurement of motor capacity
The accelerating rotarod assay (Cat. Number: 47600, Ugo Basile) consists of a base platform and a rod of 3 cm diameter and 5.7 cm length with a nonslippery surface. Mice were placed on the rod and a speed ramp from 0 to 30 rotations per minute was applied for the first 60 seconds, followed by an additional 60 seconds at the maximal speed. A cut off of 120 seconds was set after which the rotations were stopped and the animal was removed. The latency to fall from the rotating rod was observed for each animal. The apparatus was cleaned thoroughly between trials with Windex diluted in water (10%).
2.3.3. Motivated avoidance of noxious mechanical stimuli
The place avoidance task was conducted as previously described.9 The task is performed in the light/dark box described below except that a supplemental 9 W-120 V bulb was placed above the white uncovered compartment to increase aversion. The apparatus was placed on a wire mesh grid, and the 4.08 (1 g) von Frey filament was applied every 15 seconds. The left (injured paw in SNI) or right hind paw was stimulated when the animals were in the dark or in the light chamber, respectively. This paradigm creates a conflict between the aversive light compartment and aversive noxious mechanical stimulation in the dark compartment. The percentage of stimulation in the light chamber was calculated for each 5 minutes of testing during a total period of 30 minutes, corresponding to the animal's preference during each 5-minute block. Shifts in preference were calculated as the difference between the first and the last 5 minutes of testing.45 As the innate preference of a mouse is for the dark environment, the time spent in the light compartment is considered as a measure of the aversion to mechanical stimulation of the injured paw relative to aversion to the light compartment.
2.3.4. Novel object recognition task
Cognitive function was assessed using a visual nonselective, nonsustained attention task. A 2-session protocol was used for this test46: in the first session (training), mice were placed in the middle of a Plexiglas arena (50 × 50 × 30 cm) and were free to explore the entire arena for 10 minutes in the presence of 2 identical objects (the familiar objects); 2 hours later, one familiar object was replaced with a novel one and mice were placed into the box and allowed to explore for 10 minutes (testing). The time spent sniffing, licking, or physically touching the objects while facing them was defined as exploration time. The identity of the familiar and novel objects and the position of the novel object (left or right) were counterbalanced within each group to reduce possible bias due to location or object preference.8 The apparatus was cleaned thoroughly between trials with Windex diluted in water (10%).
2.4. Drug treatment
2.4.1. Chronic administration
Three months after SNI or sham surgery, animals were randomly assigned to receive saline or SAM (kind gift of Life Science Labs Supplements, LLC, Lakewood, NJ) treatment during the next 4 months. A solution of SAM (20 mg/kg) was freshly prepared each treatment day in 0.9% NaCl, and each animal received an oral administration of 8 μL. Animals received the treatment 3 times per week for 4 months. The chosen dose and the protocol used were influenced by previous studies.12,22,50
2.4.2. Acute administration—pretreatment
In another set of animals, SAM (20 or 40 mg/kg, 8 μL) was administered 24 hours + 1 hour before SNI surgery to evaluate the preventive effect on the development of pain behaviors.
2.4.3. Acute administration—posttreatment
To examine the effect of a single administration of SAM on established neuropathic pain, an additional cohort of SNI mice was treated with SAM (20 or 40 mg/kg, 8 μL) 2 months after SNI surgery and 1 hour before behavior testing (von Frey and acetone tests).
2.5. Global DNA methylation analysis
At the end of the chronic experiment, animals were sacrificed by decapitation under isoflurane anesthesia. According to the stereotaxic coordinates by Paxinos and Franklin (Paxinos G, Franklin, 2004), the frontal cortex was extracted (+1 to +3 mm anterior to Bregma, −1 to +1 mm lateral to midline, depth: 0 to −2.5 mm), frozen on dry ice, and stored at −80°C until use. The extracted cortex includes the prelimbic and intraorbital regions. Although referred to as the medial PFC in rodents, the prelimbic region corresponds functionally to the dorsolateral PFC in humans.26 The hemispheres were pooled for our study.
Genomic DNA was extracted using the DNeasy Blood & Tissue Kit Qiagen kit (Hilden, Germany). Lysate is first passed through a DNA spin column to selectively isolate DNA. Contrary to our previous work using the LUMA method (LUminometric Methylation Assay), the MethylFlashTM Methylated DNA Quantification kit (Epigentek, Farmingdale, NY) was used in this study to measure global DNA methylation. In 100 ng of genomic cortical DNA, methylated DNA was detected using capture and detection antibodies to 5-methylcytosine (5-mC) and then quantified colorimetrically by reading absorbance at 450 nm using Spectramax M2e Microplate Reader (Molecular Devices, Sunnyvale, CA). The amount of methylated DNA is proportional to the optical density intensity measured. Relative quantification was used to calculate percentage of 5-mC (%5-mC) in total DNA following the manufacturer's instructions. Each sample was run in duplicate. Three animals produced readings that were out of the range of the assay and were therefore excluded from this analysis.
The switch in DNA methylation assessment methods resulted in apparent inconsistencies in global DNA methylation. This method directly quantifies global DNA methylation by specifically measuring levels of 5-methylcytosine (5-mC), which is about 2% of total DNA in vertebrates. In contrast, the LUMA method used in our previous studies measures levels of 5-methylcytosine (5-mC) residing in the CCGG motif, resulting in numbers ranging from 50% to 60% of global DNA methylation within these CCGG motifs. Thus, although the measurement methods and resulting percentages differ between the current and previous studies, the direction of change in global DNA methylation in nerve injured animals remains consistent.
2.6. Statistical analyses
All data are expressed as mean ± SEM, and all analyses were performed using GraphPad Prism 6 (GraphPad Software Inc).
To assess the effect of chronic SAM in SNI or sham animals as a function of time, a 2-way repeated measures analysis of variance (group × time) was performed followed by the Bonferroni test for multiple comparisons. Only comparisons between saline- and SAM-treated SNI mice (*) or saline- and SAM-treated sham mice (#) are represented in the time course graphs. All other group comparisons (area under the curve [AUC0-8 and 9-16], shift in preference, novel object recognition) were analyzed using 1-way analysis of variance followed by Tukey multiple comparisons test. The novel object recognition task data were also analyzed using a 1-sided paired t test to compare exploration of the different objects (old vs new) by the same animal. Pearson correlation (1-tailed) was used to correlate mechanical thresholds during the last session (week 16) to avoid mechanical stimulation (shift in preference). Unpaired t tests were used to compare the effect of SAM on global DNA methylation. In all cases, the significance level was P < 0.05.
3. Results
3.1. Analysis of global DNA methylation in the mouse frontal cortex after chronic S-adenosylmethionine treatment
Global DNA methylation in the frontal cortices of SNI- and sham-operated and SAM- and vehicle-treated mice was assessed at the end of the experiment. Specifically, the level of 5-methylcytosine (5-mC) in the genomic DNA was analyzed 7 months after the induction of neuropathic pain and 4 months after the beginning of SAM treatment (Fig. 1). Consistent with previous reports, a trend towards decreased global DNA methylation was observed in SNI compared with sham animals (P = 0.06). After 4 months of treatment with the methyl donor, SAM, the percentage of global DNA methylation was similar in SAM-treated SNI mice compared with saline-treated sham mice (P = 0.64).
Figure 1.: Assessment of global DNA methylation in the mouse frontal cortex. A trend towards decreased global DNA methylation was observed in SNI- compared with sham-operated animals (P = 0.06, t test). After 4 months of treatment with the methyl donor SAM, global DNA methylation was similar between SAM-treated SNI mice vs saline-treated sham mice (P = 0.64, t test). n = 5 to 7 per group. PFC, prefrontal cortex; SAM, S-adenosylmethionine; SNI, spared nerve injury.
3.2. Chronic administration of the methyl donor, S-adenosylmethionine, decreased mechanical but not cold hypersensitivity in neuropathic animals
Sensitivity to mechanical stimuli was measured 3 months after SNI and sham surgery using the von Frey test. Before treatment, neuropathic mice were hypersensitive to both mechanical and cold stimuli on the injured limb compared with sham animals. Chronic SAM administration decreased SNI-induced mechanical hypersensitivity compared with saline-treated SNI mice, but had no effect on sham mice (Fig. 2A). Area under the curve analysis of mechanical sensitivity in weeks 0 to 8 vs weeks 9 to 16 of SAM treatment reflects the emergence of beneficial effects of SAM after 2 months of treatment (Fig. 2B).
Figure 2.: Effect of chronic SAM on mechanical and cold hypersensitivity and motor capacity. (A) Repeated administration of SAM (20 mg/kg, p.o., 3×/week) attenuated nerve injury–induced hypersensitivity to mechanical stimuli compared with saline but had no effect on sham-operated controls. (B) The therapeutic effects of SAM took 2 to 4 months to emerge as shown by area under the curve analysis comparing the first 2 months vs the last 2 months of treatment. (C) Spared nerve injury mice were hypersensitive to cold compared with sham; no effects of SAM were observed in either group. (D) Although saline-treated sham animals temporarily improved in the rotarod assay, no changes were observed in the sham-SAM, SNI-saline, or SNI-SAM groups. (E) Pretreatment with SAM (20 or 40 mg/kg, p.o., 1 day + 1 hour before SNI surgery) had no effect on the development of mechanical hypersensitivity in the 2 weeks postsurgery. (F) A single administration of SAM (20 or 40 mg/kg, p.o.) administered 2 months postinjury had no effect on mechanical hypersensitivity compared with saline. n = 6 to 8 (A–D) and n = 4 (E and F). *P < 0.05, **P < 0.01, SNI-saline vs SNI-SAM; #P < 0.05, ##P < 0.01, sham-saline vs sham-SAM; 2-way RM ANOVA (A, C, and D) followed by Bonferonni post hoc test (A, C–E), 2-way ANOVA followed by Bonferonni post hoc test (A, C, and D), 1-way ANOVA followed by Tukey multiple comparisons test (B and F). ANOVA, analysis of variance; RM, repeated measures; SAM, S-adenosylmethionine; SNI, spared nerve injury.
Three months after the induction of neuropathic pain, SNI animals were hypersensitive to cold compared with sham. S-adenosylmethionine had no significant effects on cold sensitivity in SNI or sham animals compared with vehicle-treated controls (Fig. 2C).
To examine the effect of SAM on motor capacity, the rotarod assay was used (Fig. 2D). No treatment effect was observed within SNI animals. In contrast, sham animals increased their performance in the rotarod test over the 16-week treatment period, with a sudden surge in sham-saline animals at 8 weeks. Although the reasons for this are unclear; it is unlikely to be a confounding factor because similar abrupt changes are not observed in other behavioural assays.
3.3. Acute administration of the methyl donor, S-adenosylmethionine, before or 2 months after nerve injury has no effect on mechanical or cold sensitivity
To determine if acute pretreatment with SAM before nerve injury would attenuate the development of neuropathic pain, an independent cohort of animals was treated with 20 or 40 mg of oral SAM 1 day and again 1 hour before SNI surgery. Pretreatment had no effect on sensitivity to mechanical (Fig. 2E) or cold (data not shown) stimuli in the first 2 weeks postinjury.
The importance of chronic exposure was confirmed in an independent set of SNI mice in which a single treatment with 20 or 40 mg/kg of oral SAM delivered 2 months after SNI surgery failed to produce any effects on mechanical (Fig. 2F) or cold (data not shown) sensitivity 1 hour after treatment.
3.4. Repeated administration of S-adenosylmethionine decreased motivated avoidance of noxious mechanical stimuli in neuropathic mice
The place avoidance assay provides a measure of the unpleasantness of noxious mechanical stimuli beyond what can be inferred from reflexive measures. The innate preference of mice to remain in a dark environment is challenged by mechanically stimulating the SNI-injured, hypersensitive, paw in a dark chamber and the uninjured, less sensitive, paw in a light chamber.35 A change in preference towards the light chamber is a measure of the aversiveness of mechanical stimulation of the injured paw (Fig. 3A). This assay was performed at the end of the chronic 4-month treatment period (7 total months after surgery). In the first 5 minutes of the assay, all groups spent between 10% and 25% of the time in the light chamber. During the 30-minute test period, saline-treated SNI animals progressively shifted their preference to the light compartment, displaying a 45% shift in preference by the end of experiment (Fig. 3B). In contrast, the shift in SAM-treated SNI animals was significantly attenuated, producing preferences similar to those observed in the sham-operated groups. The shift in preference (between first and last 5 minutes of test) was significantly lower in SNI animals treated with SAM (23.1% ± 3.6%) compared with SNI treated with saline (45.4% ± 8.2%). These data suggest that recurrent stimulation of the nerve-injured paw in SAM-treated mice was not disturbing or painful enough to induce them to leave the dark compartment. Finally, a correlation was found between mechanical sensitivity the last week of experiment (week 16) and the shift in preference (r = −0.48, P = 0.04, Fig. 3C) in SNI animals.
Figure 3.: Effect of chronic SAM on motivated pain avoidance behavior. (A) Schematic representation of the place avoidance test. (B) Spared nerve injury-saline mice developed a shift in preference for the light chamber (% last 5 minutes − initial 5 minutes) that was not observed in the SNI-SAM group. (C) Correlation between mechanical sensitivity after 4 months of SAM treatment and the shift in place preference in the pain avoidance test in SNI animals. n = 6 to 8 per group. *P < 0.05, 1-way ANOVA, followed by Tukey multiple comparisons post hoc test (B). Pearson correlation (C). ANOVA, analysis of variance; SAM, S-adenosylmethionine; SNI, spared nerve injury.
3.5. Repeated administration of S-adenosylmethionine reversed cognitive impairment in neuropathic mice
Cognitive function was assessed using a visual nonselective, nonsustained attention task. First, animals are placed in a chamber with 2 objects and allowed to explore for 10 minutes. Then, one object (old) is replaced by a new object (Fig. 4A). Saline- and SAM-treated sham animals spent more time exploring the new object compared with the familiar, old one, reflecting a preference for the new object higher than the chance level of 50% (Fig. 4B, C). In contrast, saline-treated SNI animals did not display a shift in exploration, indicating that SNI mice were impaired in this cognitive task. Chronic treatment with the methyl donor, SAM, restored the shift in attention towards the new object, suggesting that the treatment was able to reverse cognitive impairment in neuropathic mice. S-adenosylmethionine administration had no effect on sham animals in this test, and all groups showed similar overall locomotor activity during the initial and second test periods (Fig. 4D).
Figure 4.: Effect of chronic SAM on SNI-induced attention deficits. (A) Schematic representation of the novel object recognition task. (B) SNI-operated animals receiving saline solution spent the same amount of time exploring the old (O) and new (N) objects. Spared nerve injury mice receiving 4 months of SAM treatment increased their time exploring the new object. (C) The preference of the new object (%) was similar to the level of chance in vehicle-treated SNI animals. This deficit was reversed by repeated administration of SAM. (D) The difference in object exploration time between SAM- and saline-treated SNI mice was not due to locomotor impairment as the total distance travelled during this test was not different. n = 6 to 8 per group. *P < 0.05, **P < 0.01, ***P < 0.001, paired t test training vs testing (B), 1-way ANOVA followed by Tukey multiple comparisons test (C and D). ANOVA, analysis of variance; SAM, S-adenosylmethionine; SNI, spared nerve injury.
4. Discussion
The efficacy of long-term systemic treatment with the methyl donor, SAM, on the sensory, motivational, and cognitive impact of chronic neuropathic pain was assessed using the SNI model in mice. In previous studies, we demonstrated that chronic peripheral nerve injury induces decreased global DNA methylation, dysregulation of mRNA expression, and altered DNA methylation in thousands of promoters in the frontal cortex2,43; these pain symptoms and pathological changes in global methylation are attenuated by environmental enrichment.69 Structure, function, and connectivity of the PFC are altered in patients with chronic pain and can be attenuated with effective treatment.10,13,60 We therefore hypothesized that treatment with SAM would attenuate nerve injury–induced behavioural changes and restore global DNA methylation in the frontal cortex towards normal levels.
Chronic SAM treatment attenuated mechanical hypersensitivity and pain avoidance behavior and completely blocked neuropathic pain–related cognitive impairment in the novel object recognition task. Although it did not reach statistical significance (P < 0.05), a trend towards recovery of global methylation in the frontal cortex was also observed. Our results are consistent with a role for DNA methylation in mediating the sensory, affective and cognitive impact of chronic pain.
4.1. S-adenosylmethionine as a nutritional supplement
S-adenosylmethionine is marketed as a nutritional supplement for a range of conditions including osteoarthritis pain, anxiety, depression, and dementia. The study was designed to mirror the use of SAM by individuals with chronic pain: (1) chronic pain and its downstream impact were first allowed to fully develop for 3 months after nerve injury; (2) SAM was delivered orally; (3) SAM was delivered chronically 3 times a week for 4 months; and (4) sensory, affective or motivational, and cognitive assessments were included.
Beneficial effects of prolonged treatment with methyl donor diets on pain in both humans and animals were previously reported.15,30,32,63 Dietary supplements are commonly used by pain patients, with an estimated 30% of individuals with osteoarthritis using supplements to treat their condition. S-adenosylmethionine (typically 20 mg, 3× per day)30 was safe and possibly better tolerated than nonsteroidal anti–inflammatory drugs for patients with osteoarthritis.49,61 In children with abdominal pain, oral SAM has promise in reducing pain with minimal toxicity.15 Very few animal studies on the effect of SAM on pain have been published. Although Barcelo and colleagues found that intramuscular injection with 30 and 60 mg/kg of SAM for 12 weeks increased cell number and cartilage thickness in a model of osteoarthritis in rabbits,6 pain was not measured. To our knowledge, this is the first study to investigate pain symptoms or other consequences of chronic pain, including affective and cognitive deficits, in a preclinical model.
4.2. Effect of S-adenosylmethionine on motivational and cognitive comorbidities
The motivational component of pain aversion was assessed using the place avoidance task.54 This paradigm is based on a conflict between rodents' natural tendency to avoid light vs avoidance of noxious stimuli. To accomplish this, the time spent in a light vs dark box is measured over a 30-minute period during which the uninjured paw is mechanically stimulated in the light box and the injured paw is stimulated in the dark box. During the 30-minute test phase, SNI-operated mice spent gradually more time in the light area compared with controls, confirming that the injury produces motivated pain avoidance. In SNI animals treated with SAM, active avoidance of the painful stimulation was reduced to levels observed in sham-operated controls. Moreover, this shift in behavior observed in nerve-injured animals correlated with the presence of mechanical hypersensitivity detected in the von Frey test. Motor capacity and exploration are unlikely to confound this result as no injury-induced or SAM-related effects were observed in total open field exploration time. The results from the place avoidance assay add motivational and affective components to the beneficial effects of SAM on mechanical thresholds also observed in this study.
Patients with chronic pain are concerned about memory and attention deficits.21,38 Links between neuropathic pain and cognitive symptoms, including alterations of working and short-term memory,11,56 spatial and social memory,29 and attention,41 have also been reported in preclinical studies. The repeated administration of SAM reversed a SNI-induced cognitive deficit in the novel object recognition task. This is consistent with reports that SAM may be beneficial in the treatment of dementia and Alzheimer disease.47
4.3. Site of action and timing
It is now well accepted that DNA methylation is dynamically regulated in the adult nervous system and may be important in synaptic plasticity, memory formation,57 and memory of pain.2 In the last decade, several studies investigated DNA methylation along the pain pathway, specifically in dorsal root ganglia, the dorsal horn, and brain after nerve injury.43,51,53,66,70–72 Although DNA methylation increased in dorsal horn 14 days after chronic construction injury,71 it decreased in the frontal cortex and amygdala 6 months after SNI.66 These differences may be due to the existence of divergent regulatory mechanisms in different tissues or variations in the pain models and time points investigated. What is clear is that epigenetic regulation occurs throughout the neuroaxis in many preclinical pain models,18,23,39 and DNA methylation has been linked to sensitivity to noxious stimuli in humans.7
The rationale for this study was driven by previous observations that SNI induces decreases in global DNA methylation in frontal cortex and that this decrease can be reversed by pain-reducing environmental enrichment.66,69 In this study, SNI resulted in a trend towards decreased DNA methylation in the frontal cortex (P = 0.06) compared with sham-operated control animals that was absent after treatment with SAM (P = 0.64). These results are consistent with other data demonstrating that systemic SAM can modulate global DNA methylation within the central nervous system.22 Although these results are consistent with a role for cortical DNA methylation in the therapeutic effects of SAM, additional experiments are required to address that hypothesis directly.
Beneficial effects on mechanical sensory thresholds were not observed until 4 to 8 weeks into the treatment and grew in magnitude with time. S-adenosylmethionine was not initiated until 3 months after nerve injury to ensure that the impact of chronic pain on the central nervous system had time to fully develop. It is reasonable to propose that weeks to months of treatment are required to reverse established pathological changes. Consistent with this is the observation that beneficial effects of environmental enrichment with voluntary running in the same model did not appear until 2 months.66,69 In our study, when SAM was administrated before the surgery or as a single dose 2 months post-SNI, no effect was observed on mechanical or cold hypersensitivity. Nevertheless, additional chronic experiments are needed to examine potential beneficial preventive effects of SAM when administrated chronically months before surgery or at the time of the induction of neuropathic pain. Thus, although analgesic treatments may have immediate efficacy by reducing nociceptive signaling, disease-modifying treatments like physical activity or methylome modifications may take longer to emerge.
An alternative hypothesis regarding the timing relates to the time course of nerve injury–related pathology. In a study using the SNI model in rats, it took 3 to 4 months for (1) anxiety-like symptoms to occur and (2) for measurable reductions in gray matter to be detected.60 Thus, the entire system may be reset several months after an injury compared with before or a few weeks after injury. It is currently unknown if changes observed in DNA methylation or other epigenetic mechanisms occur immediately after injury vs weeks vs months, nor is it known when in this progression maximum therapeutic benefit can be realized. It is therefore possible that the underlying mechanisms _targeted by SAM may not exist until 5 months after injury. The next step is the exploration of the timing of pain-related brain pathology and how it relates to the emergence and sensitivity of different sensory, motivational and affective, and cognitive symptoms.
4.4. Mechanism of action
Although the therapeutic benefits of SAM observed here are consistent with its role as a methyl donor for DNA methyltransferases, SAM is also a substrate for other pain-related enzymes. For example, catechol O-methyltransferase (COMT) is an enzyme that has been implicated in the perception of mechanical, thermal, and inflammatory pain in both humans and rodents. Catechol O-methyltransferase is responsible for the degradation of catechol-containing compounds including dopamine, epinephrine, and norepinepherine. Catecholamines modulate pain perception, and increases in COMT are associated with lower perceived pain intensity.19,20,59 As a methyl donor, SAM augments COMT activity,62 which would be predicted to decrease pain sensitivity. Moreover, a recent article showed that serotonin is able to inhibit COMT by actively competing with SAM within the catalytic site of COMT. This serotonergic inhibition of COMT contributes to pain hypersensitivity.68 The results of this study suggest additional strategies for COMT-dependent pain modulation.
5. Summary
Chronic systemic administration of SAM reduced peripheral nerve injury–induced mechanical hypersensitivity, pain avoidance behavior, and cognitive impairment. Moreover, the SNI-induced reduction in global DNA methylation in frontal cortex was partially reversed by chronic administration of SAM. These findings support the hypothesis that DNA methylation is functionally involved in chronic pain and have possible implications for the development of future pain treatment strategies.
Conflict of interest statement
The authors have no conflicts of interest and funding sources had no influence on the experimental design, data analysis, or in the preparation of the manuscript.
Supported by a grant from Pfizer Canada to L. S. Stone and M. Szyf, a grant from the Canadian Institutes for Health Research to L. S. Stone, M. Szyf, and M. Millecamps and a Louise and Alan Edwards Foundation Postdoctoral Fellowship to S. Grégoire.
Acknowledgements
The authors thank Dr. Renaud Massart, Dr. Maral Tajerian, and Dr. Sebastian Alvarado for their contributions and Life Science Labs Supplements, LLC for the S-adenosylmethionine (SAM).
References
[1]. Alvarado S, Tajerian M, Millecamps M, Suderman M, Stone LS, Szyf M. Peripheral nerve injury is accompanied by chronic transcriptome-wide changes in the mouse prefrontal cortex. Mol Pain 2013;9:21.
[2]. Alvarado S, Tajerian M, Suderman M, Machnes Z, Pierfelice S, Millecamps M, Stone LS, Szyf M. An epigenetic hypothesis for the genomic memory of pain. Front Cell Neurosci 2015;9:88.
[3]. Apkarian AV, Sosa Y, Sonty S, Levy RM, Harden RN, Parrish TB, Gitelman DR. Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. J Neurosci 2004;24:10410–15.
[4]. Auvray M, Myin E, Spence C. The sensory-discriminative and affective-motivational aspects of pain. Neurosci Biobehav Rev 2010;34:214–23.
[5]. Baliki MN, Chang PC, Baria AT, Centeno MV, Apkarian AV. Resting-sate functional reorganization of the rat limbic system following neuropathic injury. Sci Rep 2014;4:6186.
[6]. Barcelo HA, Wiemeyer JC, Sagasta CL, Macias M, Barreira JC. Effect of S-adenosylmethionine on experimental osteoarthritis in rabbits. Am J Med 1987;83:55–9.
[7]. Bell JT, Loomis AK, Butcher LM, Gao F, Zhang B, Hyde CL, Sun J, Wu H, Ward K, Harris J, Scollen S, Davies MN, Schalkwyk LC, Mill J, Williams FM, Li N, Deloukas P, Beck S, McMahon SB, Wang J, John SL, Spector TD. Differential methylation of the TRPA1 promoter in pain sensitivity. Nat Commun 2014;5:2978.
[8]. Bevins RA, Besheer J. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study “recognition memory”. Nat Protoc 2006;1:1306–11.
[9]. Burke NN, Finn DP, Roche M. Chronic administration of amitriptyline differentially alters neuropathic pain-related behaviour in the presence and absence of a depressive-like phenotype. Behav Brain Res 2015;278:193–201.
[10]. Bushnell MC, Ceko M, Low LA. Cognitive and emotional control of pain and its disruption in chronic pain. Nat Rev Neurosci 2013;14:502–11.
[11]. Cardoso-Cruz H, Lima D, Galhardo V. Impaired spatial memory performance in a rat model of neuropathic pain is associated with reduced hippocampus-prefrontal cortex connectivity. J Neurosci 2013;33:2465–80.
[12]. Carrasco R, Perez-Mateo M, Gutierrez A, Esteban A, Mayol MJ, Caturla J, Ortiz P. Effect of different doses of S-adenosyl-L-methionine on paracetamol hepatotoxicity in a mouse model. Methods Find Exp Clin Pharmacol 2000;22:737–40.
[13]. Ceko M, Shir Y, Ouellet JA, Ware MA, Stone LS, Seminowicz DA. Partial recovery of abnormal insula and dorsolateral prefrontal connectivity to cognitive networks in chronic low back pain after treatment. Hum Brain Mapp 2015;36:2075–92.
[14]. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53:55–63.
[15]. Choi LJ, Huang JS. A pilot study of S-adenosylmethionine in treatment of functional abdominal pain in children. Altern Ther Health Med 2013;19:61–4.
[16]. Choi Y, Yoon YW, Na HS, Kim SH, Chung JM. Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain. PAIN 1994;59:369–76.
[17]. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. PAIN 2000;87:149–58.
[18]. Descalzi G, Ikegami D, Ushijima T, Nestler EJ, Zachariou V, Narita M. Epigenetic mechanisms of chronic pain. Trends Neurosci 2015;38:237–46.
[19]. Diatchenko L, Nackley AG, Slade GD, Bhalang K, Belfer I, Max MB, Goldman D, Maixner W. Catechol-O-methyltransferase gene polymorphisms are associated with multiple pain-evoking stimuli. PAIN 2006;125:216–24.
[20]. Diatchenko L, Slade GD, Nackley AG, Bhalang K, Sigurdsson A, Belfer I, Goldman D, Xu K, Shabalina SA, Shagin D, Max MB, Makarov SS, Maixner W. Genetic basis for individual variations in pain perception and the development of a chronic pain condition. Hum Mol Genet 2005;14:135–43.
[21]. Dick BD, Rashiq S. Disruption of attention and working memory traces in individuals with chronic pain. Anesth Analg 2007;104:1223–9, tables of contents.
[22]. Do Carmo S, Hanzel CE, Jacobs ML, Machnes Z, Iulita MF, Yang J, Yu L, Ducatenzeiler A, Danik M, Breuillaud LS, Bennett DA, Szyf M, Cuello AC. Rescue of Early bace-1 and global DNA Demethylation by S-Adenosylmethionine reduces Amyloid pathology and Improves cognition in an Alzheimer's model. Sci Rep 2016;6:34051.
[23]. Doehring A, Geisslinger G, Lotsch J. Epigenetics in pain and analgesia: an imminent research field. Eur J Pain 2011;15:11–16.
[24]. Dong E, Agis-Balboa RC, Simonini MV, Grayson DR, Costa E, Guidotti A. Reelin and glutamic acid decarboxylase67 promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia. Proc Natl Acad Sci U S A 2005;102:12578–83.
[25]. Dousset V, Maud M, Legoff M, Radat F, Brochet B, Dartigues JF, Kurth T. Probable medications overuse headaches: validation of a brief easy-to-use screening tool in a headache centre. J Headache Pain 2013;14:81.
[26]. Farovik A, Dupont LM, Arce M, Eichenbaum H. Medial prefrontal cortex supports recollection, but not familiarity, in the rat. J Neurosci 2008;28:13428–34.
[27]. Geranton SM, Tochiki KK. Regulation of gene expression and pain states by epigenetic mechanisms. Prog Mol Biol Transl Sci 2015;131:147–83.
[28]. Global Burden of Disease Study C. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;386:743–800.
[29]. Gregoire S, Michaud V, Chapuy E, Eschalier A, Ardid D. Study of emotional and cognitive impairments in mononeuropathic rats: effect of duloxetine and gabapentin. PAIN 2012;153:1657–63.
[30]. Gregory PJ, Sperry M, Wilson AF. Dietary supplements for osteoarthritis. Am Fam Physician 2008;77:177–84.
[31]. Hollingsworth JW, Maruoka S, Boon K, Garantziotis S, Li Z, Tomfohr J, Bailey N, Potts EN, Whitehead G, Brass DM, Schwartz DA. In utero supplementation with methyl donors enhances allergic airway disease in mice. J Clin Invest 2008;118:3462–9.
[32]. Hosea Blewett HJ. Exploring the mechanisms behind S-adenosylmethionine (SAMe) in the treatment of osteoarthritis. Crit Rev Food Sci Nutr 2008;48:458–63.
[33]. Kim J, Lee EY, Koh EM, Cha HS, Yoo B, Lee CK, Lee YJ, Ryu H, Lee KH, Song YW. Comparative clinical trial of S-adenosylmethionine versus nabumetone for the treatment of knee osteoarthritis: an 8-week, multicenter, randomized, double-blind, double-dummy, Phase IV study in Korean patients. Clin Ther 2009;31:2860–72.
[34]. Kiritoshi T, Ji G, Neugebauer V. Rescue of impaired mGluR5-driven Endocannabinoid signaling restores prefrontal cortical Output to inhibit pain in Arthritic rats. J Neurosci 2016;36:837–50.
[35]. LaBuda CJ, Fuchs PN. A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats. Exp Neurol 2000;163:490–4.
[36]. Lan J, Hua S, He X, Zhang Y. DNA methyltransferases and methyl-binding proteins of mammals. Acta Biochim Biophys Sin (Shanghai) 2010;42:243–52.
[37]. Lee M, Manders TR, Eberle SE, Su C, D'Amour J, Yang R, Lin HY, Deisseroth K, Froemke RC, Wang J. Activation of corticostriatal circuitry relieves chronic neuropathic pain. J Neurosci 2015;35:5247–59.
[38]. Legrain V, Damme SV, Eccleston C, Davis KD, Seminowicz DA, Crombez G. A neurocognitive model of attention to pain: behavioral and neuroimaging evidence. PAIN 2009;144:230–2.
[39]. Liang L, Lutz BM, Bekker A, Tao YX. Epigenetic regulation of chronic pain. Epigenomics 2015;7:235–45.
[40]. Ligon CO, Moloney RD, Greenwood-van Meerveld B. _targeting epigenetic mechanisms for chronic pain: a valid approach for the development of novel therapeutics. J Pharmacol Exp Ther 2016;357:84–93.
[41]. Low LA, Millecamps M, Seminowicz DA, Naso L, Thompson SJ, Stone LS, Bushnell MC. Nerve injury causes long-term attentional deficits in rats. Neurosci Lett 2012;529:103–7.
[42]. MacDonald JL, Roskams AJ. Epigenetic regulation of nervous system development by DNA methylation and histone deacetylation. Prog Neurobiol 2009;88:170–83.
[43]. Massart R, Dymov S, Millecamps M, Suderman M, Gregoire S, Koenigs K, Alvarado S, Tajerian M, Stone LS, Szyf M. Overlapping signatures of chronic pain in the DNA methylation landscape of prefrontal cortex and peripheral T. Sci Rep 2016;6:19615.
[44]. McWilliams LA, Cox BJ, Enns MW. Mood and anxiety disorders associated with chronic pain: an examination in a nationally representative sample. PAIN 2003;106:127–33.
[45]. Millecamps M, Centeno MV, Berra HH, Rudick CN, Lavarello S, Tkatch T, Apkarian AV. D-cycloserine reduces neuropathic pain behavior through limbic NMDA-mediated circuitry. PAIN 2007;132:108–23.
[46]. Millecamps M, Etienne M, Jourdan D, Eschalier A, Ardid D. Decrease in non-selective, non-sustained attention induced by a chronic visceral inflammatory state as a new pain evaluation in rats. PAIN 2004;109:214–24.
[47]. Montgomery SE, Sepehry AA, Wangsgaard JD, Koenig JE. The effect of S-adenosylmethionine on cognitive performance in mice: an animal model meta-analysis. PLoS One 2014;9:e107756.
[48]. Moriarty O, McGuire BE, Finn DP. The effect of pain on cognitive function: a review of clinical and preclinical research. Prog Neurobiol 2011;93:385–404.
[49]. Muller-Fassbender H. Double-blind clinical trial of S-adenosylmethionine versus ibuprofen in the treatment of osteoarthritis. Am J Med 1987;83:81–3.
[50]. Palomero J, Galan AI, Munoz ME, Gonzalez-Gallego J, Tunon MJ, Jimenez R. S-adenosylmethionine protects against intrabiliary glutathione degradation induced by long-term administration of cyclosporin A in the rat. Toxicology 2004;201:239–45.
[51]. Pan Z, Zhu LJ, Li YQ, Hao LY, Yin C, Yang JX, Guo Y, Zhang S, Hua L, Xue ZY, Zhang H, Cao JL. Epigenetic modification of spinal miR-219 expression regulates chronic inflammation pain by _targeting CaMKIIgamma. J Neurosci 2014;34:9476–83.
[52]. Papakostas GI. Evidence for S-adenosyl-L-methionine (SAM-e) for the treatment of major depressive disorder. J Clin Psychiatry 2009;70(suppl 5):18–22.
[53]. Pollema-Mays SL, Centeno MV, Apkarian AV, Martina M. Expression of DNA methyltransferases in adult dorsal root ganglia is cell-type specific and up regulated in a rodent model of neuropathic pain. Front Cell Neurosci 2014;8:217.
[54]. Refsgaard LK, Hoffmann-Petersen J, Sahlholt M, Pickering DS, Andreasen JT. Modelling affective pain in mice: effects of inflammatory hypersensitivity on place escape/avoidance behaviour, anxiety and hedonic state. J Neurosci Methods 2016;262:85–92.
[55]. Remington R, Chan A, Paskavitz J, Shea TB. Efficacy of a vitamin/nutriceutical formulation for moderate-stage to later-stage Alzheimer's disease: a placebo-controlled pilot study. Am J Alzheimers Dis Other Demen 2009;24:27–33.
[56]. Ren WJ, Liu Y, Zhou LJ, Li W, Zhong Y, Pang RP, Xin WJ, Wei XH, Wang J, Zhu HQ, Wu CY, Qin ZH, Liu G, Liu XG. Peripheral nerve injury leads to working memory deficits and dysfunction of the hippocampus by upregulation of TNF-alpha in rodents. Neuropsychopharmacology 2011;36:979–92.
[57]. Roth TL, Sweatt JD. Regulation of chromatin structure in memory formation. Curr Opin Neurobiol 2009;19:336–42.
[58]. Schaible TD, Harris RA, Dowd SE, Smith CW, Kellermayer R. Maternal methyl-donor supplementation induces prolonged murine offspring colitis susceptibility in association with mucosal epigenetic and microbiomic changes. Hum Mol Genet 2011;20:1687–96.
[59]. Segall SK, Nackley AG, Diatchenko L, Lariviere WR, Lu X, Marron JS, Grabowski-Boase L, Walker JR, Slade G, Gauthier J, Bailey JS, Steffy BM, Maynard TM, Tarantino LM, Wiltshire T. Comt1 genotype and expression predicts anxiety and nociceptive sensitivity in inbred strains of mice. Genes Brain Behav 2010;9:933–46.
[60]. Seminowicz DA, Laferriere AL, Millecamps M, Yu JS, Coderre TJ, Bushnell MC. MRI structural brain changes associated with sensory and emotional function in a rat model of long-term neuropathic pain. Neuroimage 2009;47:1007–14.
[61]. Soeken KL, Lee WL, Bausell RB, Agelli M, Berman BM. Safety and efficacy of S-adenosylmethionine (SAMe) for osteoarthritis. J Fam Pract 2002;51:425–30.
[62]. Strous RD, Ritsner MS, Adler S, Ratner Y, Maayan R, Kotler M, Lachman H, Weizman A. Improvement of aggressive behavior and quality of life impairment following S-adenosyl-methionine (SAM-e) augmentation in schizophrenia. Eur Neuropsychopharmacol 2009;19:14–22.
[63]. Sun Y, Liang D, Sahbaie P, Clark JD. Effects of methyl donor diets on incisional pain in mice. PLoS One 2013;8:e77881.
[64]. Suzuki MM, Bird A. DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 2008;9:465–76.
[65]. Tajerian M, Alvarado S, Millecamps M, Dashwood T, Anderson KM, Haglund L, Ouellet J, Szyf M, Stone LS. DNA methylation of SPARC and chronic low back pain. Mol Pain 2011;7:65.
[66]. Tajerian M, Alvarado S, Millecamps M, Vachon P, Crosby C, Bushnell MC, Szyf M, Stone LS. Peripheral nerve injury is associated with chronic, reversible changes in global DNA methylation in the mouse prefrontal cortex. PLoS One 2013;8:e55259.
[67]. Tracey I, Bushnell MC. How neuroimaging studies have challenged us to rethink: is chronic pain a disease? J Pain 2009;10:1113–20.
[68]. Tsao D, Wieskopf JS, Rashid N, Sorge RE, Redler RL, Segall SK, Mogil JS, Maixner W, Dokholyan NV, Diatchenko L. Serotonin-induced hypersensitivity via inhibition of catechol O-methyltransferase activity. Mol Pain 2012;8:25.
[69]. Vachon P, Millecamps M, Low L, Thompsosn SJ, Pailleux F, Beaudry F, Bushnell CM, Stone LS. Alleviation of chronic neuropathic pain by environmental enrichment in mice well after the establishment of chronic pain. Behav Brain Funct 2013;9:22.
[70]. Wang Y, Lin ZP, Zheng HZ, Zhang S, Zhang ZL, Chen Y, You YS, Yang MH. Abnormal DNA methylation in the lumbar spinal cord following chronic constriction injury in rats. Neurosci Lett 2016;610:1–5.
[71]. Wang Y, Liu C, Guo QL, Yan JQ, Zhu XY, Huang CS, Zou WY. Intrathecal 5-azacytidine inhibits global DNA methylation and methyl- CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury. Brain Res 2011;1418:64–9.
[72]. Zhang HH, Hu J, Zhou YL, Qin X, Song ZY, Yang PP, Hu S, Jiang X, Xu GY. Promoted Interaction of Nuclear factor-kappaB with Demethylated Purinergic P2X3 Receptor gene contributes to neuropathic pain in rats with Diabetes. Diabetes 2015;64:4272–84.
[73]. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. PAIN 1983;16:109–10.
[74]. Zimmermann M. Ethical considerations in relation to pain in animal experimentation. Acta Physiol Scand Suppl 1986;554:221–33.
